The present invention is related to post chromatographic column fluid flow and in particular to a fluid splitting device to provide an interface between the liquid chromatographic column and the subsequent detection and analysis equipment.
High performance liquid chromatography-mass spectrometry (LC-MS) is a widely applied technique with a capacity for fast and sensitive characterization and quantification of pharmaceutical compounds and their metabolites. The analysis of these agents in complex biological fluids such as plasma, urine, bile and tissue homogenates, for the determination of pharmacokinetic parameters and metabolic pathways is a crucial step in the drug development process. The inherent specificity of mass detection coupled with the compound separation afforded by liquid chromatography has contributed to increased analytical productivity in the area of quantitative analysis by reducing the need for extensive sample preparation. In addition, the unique combination of detection sensitivity and information content has made LC-MS an essential tool in the determination of metabolic pathways. Such tools have resulted in reduced assay development times, reduced analysis times, and improved detection limits. Currently, several thousand quantitative assays can be carried out in a single day through the use of 96, 384 and higher well plate technology and tandem mass spectrometric (MS-MS) techniques. Furthermore, development in software applications has enabled the automated quantitative and qualitative characterization of drugs and metabolites, thus alleviating the bottleneck in data processing generated by increased sample throughput. The use of LC-MS based methodologies has become popular and widespread in the pharmaceutical industry. While these analytical techniques have provided outstanding results in recent years, there have been other problems associated with them.
For example, efforts to increase sample throughput have placed huge demands on analytical instrumentation to obtain near error-free measurements over long periods of time. In many instances, matrix components present in these samples are responsible for analysis failures and errors. These matrix components foul instrumentation and interfere with the detection process in mass spectrometry. Because ion transmission through the MS analyzer generally depends upon mass and mass-to-charge ratios and not on analyte structural features, problems with analyte detection have often been attributed to processes in the electrospray ionization region of the mass spectrometer, such as ionization suppression by the matrix components. Although the mechanism by which matrix components suppress the analyte signal is not fully understood, many components in biological fluids, such as salts, bile acids and other compounds, may exist in very large relative abundance to the desired analyte. These components also may have very high ionization efficiencies (which will mask the desired analyte signal) or high surface activities that can reduce the analyte response and compromise the quality of the analytical measurements. In some cases, matrix components can cause ionization suppression to such an extent that major metabolites are rendered undetectable by MS.
These problems have resulted in the need for time-consuming sample preparation methods in order to remove a portion of the matrix components. However, such methods are often inadequate. For example, solid phase extraction methods are only moderately successful at limiting suppression effects since they rely on large differences in chromatographic behavior for matrix removal. Matrix components that remain following solid phase extraction cleanup typically have chromatographic behavior similar to that of the analytes. As a result, these components are likely to coelute with the analyte in LC-MS and continue to cause ion suppression and inaccurate composition results in MS.
It has been shown that reducing the electrospray ion flow rate down to the nanoliter per minute range leads to improvements in desolvation, ionization and ion transfer efficiencies over conventional electrospray ionization flows. In order to exploit these lower flow benefits, it becomes necessary to either utilize capillary columns or split the effluent from a large bore chromatographic column before it enters the mass spectrometer. The use of capillary columns suffers from many limitations, including lower mass loading and contamination due to matrix components which can lead to rapid deterioration in column performance. Larger diameter columns do not suffer from such drawbacks and as such offer more rugged and reproducible separations. Therefore, in order to take advantage of the benefits of each stage of an LC-MS analysis, an integrated system would require the effluent from a large bore LC column be split so that a reduced flow rate is introduced into the MS. A common technique used to reduce the flow rate of the column effluent is the use of a xe2x80x9cTxe2x80x9d configuration fluid divider. Typically, in these dividers the effluent enters from one side and exits through the two outflow dividers. The ratio of the output flow rates is determined by the outflow dividers"" flow resistances. These flow rates may be finely adjusted by providing a restriction valve on one arm of the divider. By adjusting the restriction valve, backpressure can be increased or decreased to adjust the flow rate of the other output arm. These dividers, however, tend to reduce the sensitivity and resolution of the chromatographic analysis due to band broadening caused by turbulence and/or mixing at the xe2x80x9csplit pointxe2x80x9d within the divider. In addition, current splitting devices contain a relatively large fluid volume between the fluid split point and the detection device. In general, the larger the volume introduced in the fluid path, the larger the impact on the chromatographic bands due to broadening.
Therefore, it would be advantageous to provide a fluid splitter or divider system that can be coupled to a standard size chromatographic column and in which turbulence at the split point has been minimized.
The present invention is directed to a fluid splitting device that provides an accurate output fluid flow rate in which the turbulence and mixing at the split point are substantially minimized. A fluid flow splitting device in accordance with the present invention includes an outer fluid conduit that has first and second ends, an exterior surface, a fluid inlet coupled to the incoming fluid and a fluid outlet spaced apart from the fluid inlet. The fluid splitting device further includes an inner fluid conduit that is coaxially disposed within the outer fluid conduit. The inner fluid conduit has a fluid input end in fluid communication with the fluid inlet of the outer fluid conduit, wherein the fluid input end forms a fluid split point at which a portion of the input fluid is diverted so that it flows into the inner fluid conduit. The inner fluid conduit also has an output end that extends beyond the exterior surface of the outer fluid conduit and provides an output fluid flow at an output fluid flow rate, which directly interfaces with the detection method. Advantageously, this minimizes the extra-column effects by minimizing the fluid volume between the split point and the detector. The input end of the inner fluid conduit, i.e., the split point, is interposed between the fluid inlet and the fluid outlet of the outer fluid conduit. The positioning of the fluid split point away from the fluid outlet helps to ensure that the fluid input end of the inner fluid conduit is substantially free from turbulence from the fluid outlet. The portion of the incoming fluid that flows into the fluid input end of the inner fluid conduit forms the output fluid of the device and has a flow rate that is less than the input flow rate. Use of an inner fluid conduit of minimal volume (in the low nanoliter range) enables optimal chromatographic performance. The remaining input fluid leaves the outer fluid conduit via the fluid outlet as a waste fluid at a waste fluid flow rate. The output fluid flow rate can be adjusted by adjusting the relative dimensions of the inner and outer fluid conduit. If desired, a variable flow resistor such as a restriction valve or a predetermined length of fluid conduit having a predetermined flow resistance can be coupled to the fluid outlet of the outer fluid conduit and used to adjust the waste fluid flow rate, thereby adjusting the output fluid flow rate.
Other features, aspects, and advantages of the present invention will be apparent from the Detailed Description of the Invention in conjunction with the drawing.